Define gene expression
The process by which the information encoded in a gene is turned into a function. This mostly occurs via the transcription of RNA molecules that code for proteins or non-coding RNA molecules that serve other functions
What is the use of mRNA expression measurements
- Estimate environmental or genetic effects of phenotypes
- Find differences in gene expression that could explain differences in phenotypes
What do you need to complete a PCR?
- Template DNA
- Heat stable polymerase
- Water
- Oligonucleotides (primer)
- Precise thermocycling
- Nucleotides
What is step 1 of PCR
Denaturation
- water with DNA in there
- 90 degrees
- 1 min
- DNA splits into single strands
What is step 2 of PCR
Annealing
- Cool whole thing down
- Add primers that bind to complementary DNA
- 54 degrees
- 45 seconds
What is step 3 of PCR
Extension
- Heat up again
- Add nucleotides
- 72 degrees
- 2 mins
At what cycle of PCR do you get target DNA
not until the 3rd cycle.
This is because you dont want any DNA with longer tails than the initial DNA
What makes a good primer?
- Unique in the genome
- lack of secondary binding sites
- Tm = 52-65 degrees
- Absence of dimerization capability
- Low specific binding at the 3’ end
Describe uniqueness in terms of PCR primers
- There shall be only one target site in the template DNA where the primer binds
- There shall be no annealing site in possible contaminant sources
Describe why the length of the primer is important
- effects the uniqueness and the Tm of the primer
- Longer the primer the more chance it is unique
- HOWEVER - the longer the primer the higher the annealing temperature - bad
- Typically 17-28 bases long
Why is base composition important in a primer
- High GC content means binds strong, this is because GC=3H bonds and AT=2H bonds
- It effects the hybridisation specificity and annealing temp
- There should be no long AT/GC regions
- Usually GC content = 50-60%
Why is melting temperature important in primers
- Tm is the temp at which half the DNA strands are single stranded and half are double stranded
- High GC=High Tm because of increased H bonds
- Shorter than 13 bp = Tm = (wA x xT)x2 + (yG + zC)x4
- Longer than 13 bp = Tm = 64.9 + 41 (yG + zC - 16.4)/(wA+xT+yG+zC)
Why is annealing temperature important in primers
- The temperature at which primers anneal to the template DNA
- Tanneal = Tm-4
Why are primer secondary structures bad
- If primers anneal to themselves or anneal to each other rather than the template, the PCR efficiency will decrease
- Hairpin, Delf-dimer and dimer
- Can be harmless if annealing temp doesn’t allow them to form
What is the max difference in annealing temp between the forward and reverse primer?
3 degrees
What are the main points to remember in primer design
- Unique
- Length - 17-28 bases
- Base composition - GC- 50-60%
- High stability 5’ end, low stability 3’ end
- Tm = 55-80 degrease
- Primer sets to anneal 2-3 degrees of each other
- Minimize secondary structure
What are the primer design softwares?
- Ensembl - sequence data
- Primer3 - Primer design
- Repeat masker - Mask repeat sequences
- PrimerBLAST - check for off target effects
Briefly describe rt-qPCR
- Design 2 primers for gene of interest
- Design 2 primers for the housekeeping gene
- Relative expression of gene x vs hk gene
- small no of genes
- large no of samples
- very cheap
- all values are relative
What is the housekeeper gene?
- Genes that encode for proteins that are essential for cellular function
- Often remains constant under most experimental conditions
- Common housekeepers:
- ACTB
- GAPDH
-rRNA - UBC
What are the steps for the analysis of qPCR
- Compute the mean of the technical replicates
- Compute delta CT
- Compute mean delta CT for the control
- Compute delta delta CT
- Compute 2-delta delta CT
How do you know that output of qPCR is reliable?
- delta CT values reliable when
<0.3 away from the median of the group
What do you do if the control is not reliable
Use the geometric mean
If there is a single outlier remove the offending replicate
Otherwise redo the experiment
What are micro-arrays?
- collection of microscopic DNA spots of a slide
- Each spot have DNA known as probes or reporters
- Target fluorophore - labelled target cDNA is hybridised in the arrays
- Hybridisation is detected by intensity, giving a relative abundance of target sequence
Describe the micro-array workflow
Sample
Purification
RT - reverse transcriptase to get only DNA not RNA
Coupling
Hybridization and washes
scanning
normalization
analysis
